Didemnin B is a macrocyclic depsipeptide isolated from a species of marine tunicate. Didemnin B exhibits potent anti-viral, immunosuppressive, and anti-tumor activities in vitro and in vivo, and was the first marine natural product to enter clinical testing against human cancers (Li et al., 1992, Studies in Natural Products Chemistry, 10:241-302; Sakai et al., 1996, J. Med. Chem. 39:2819-2834; Wipf, 1995, Chem. Rev. 95:2115-2134). Didemnin B is a didemnin, a family of compounds which potently inhibit protein synthesis and cell cycle progression, and induce more rapid apoptosis than any other natural products that has been isolated to date (Grubb et al., 1995, Biochem. Biophys, Res. Commun. 215:1130-1136; Johnson et al., 1996, FEBS Lett. 383:1-5; Johnson et al., 1999, Immunol. Cell Biol. 77:242-248; Johnson et al., 1999, J. Cell. Biochem. 72:269-278). Other members of this family of compounds, including didemnin M and dehydrodidemnin B, exhibit cytotoxic and cytostatic effects as well.
Tamandarin A (also designated {(2S)HIV2}didemnin B) is a naturally occurring didemnin analog which has recently been isolated from a marine tunicate. Tamandarin A exhibits biological activity which is analogous to the activities exhibited didemnin B. For example, tamandarin A is a potent inhibitor of protein synthesis, cell growth, and tumorigenesis. Tamandarin A exhibits greater in vitro activity against pancreatic carcinoma than does didemnin B (Liang et al., 1999, Org. Lett. 1: 1319-1322). A significant limitation on use of tamandarin A, either for research or for practical applications, is the limited supply of tamandarin A that is available from natural sources and the difficulty and expense of isolating this product. A need exists for a method of synthesizing tamandarin A and other didemnin analogs (including dehydrodidemnin analogs).
Despite the potency of didemnin B in isolated studies, its clinical effectiveness is hampered by side effects associated with therapeutic doses of the compound. As with many anti-proliferative agents, didemnin B exhibits a relatively narrow therapeutic window. Although didemnin M and dehydrodidemnin B exhibit improved therapeutic potential, relative to didemnin B, a need still exists for anti-proliferative agents which exhibit less toxicity at a therapeutic dose (i.e. didemnin analogs having a greater therapeutic index).
The present invention satisfies the needs set forth above.
The invention relates to a composition comprising a didemnin analog having the structure of formula I 
In formula I, R1 is selected from the group consisting of
xe2x80x94H,
xe2x80x94(tert-butyloxycarbonyl),
xe2x80x94leucine,
xe2x80x94(N-methyl)leucine,
xe2x80x94(N-methyl)leucine-proline,
xe2x80x94(N-methyl)leucine-proline-lactate,
xe2x80x94(N-methyl)leucine-proline-pyruvate,
xe2x80x94(N-methyl)leucine-proline-lactate-(a first fluorophore),
xe2x80x94(N-methyl)leucine-proline-lactate-glutamine-pyroglutamate,
xe2x80x94(N-methyl)leucine-proline-lactate-glutamine-cyclopentanoate,
xe2x80x94(N-methyl)leucine-proline-alanine-leucine-pyroglutamate, and
xe2x80x94(N-methyl)leucine-proline-(N-methyl-alanine)leucine-pyroglutamate.
R2 and R3 in formula I, can be separate moieties or they can, together, be a single moiety. When R2 and R3 are separate moieties, R3 is either a methyl group or a hydride radical and R2 is selected from the group consisting of an isoleucine side chain, a valine side chain, an alanine side chain, a norleucine side chain, a norvaline side chain, leucine side chain, a histidine side chain, a tryptophan side chain, an arginine side chain, a lysine side chain, a second fluorophore, and a substituent having the structure of formula III 
When R2 and R3 are, together, a single substituent, this substituent has the structure of formula IV 
A In formulas III and IV, each of R5, R6, R7, R8, and R9 is independently selected from the group consisting of xe2x80x94H, xe2x80x94OH, xe2x80x94OCH3, xe2x80x94CO(C6H5), xe2x80x94Br, xe2x80x94I, xe2x80x94F, xe2x80x94Cl, CH3, and xe2x80x94C2H5.
R4 in formula I is either an isoleucine side chain or a valine side chain. Also, in formula I, X is either xe2x80x94Oxe2x80x94 or xe2x80x94(NH)xe2x80x94, Y is either a hydride radical or a hydroxyl protecting group, and R10 is either a leucine side chain or a lysine side chain. The didemnin analog is an analog other than tamandarin A (i.e. {(2S)Hiv2}didemnin B). In one embodiment, every proline or lactate moiety that is present in R1 exhibits (S) stereochemistry. In another, every moiety capable of exhibiting stereochemistry in R1 is present in its naturally occurring form (i.e. the (S) form for amino acid residues and lactate. It is believed that cyclopentanoate occurs naturally in an (S) stereochemistry.
Examples of didemnin analogs that are included in the invention are compound 103, compound 104, compound 105, compound 106, compound 107, compound 108, compound 109, compound 110, compound 115, compound 116, compound 117, compound 118, compound 119, compound 120, compound 121, compound 122, compound 123, compound 124, compound 125, compound 126, compound 127, compound 128, compound 129, compound 130, compound 133, compound 134, compound 136, compound 137, compound 139, compound 141, compound 142, compound 143.
In one embodiment, the didemnin analog has a photoreactive substituent, such as an R2 moiety having the structure 
In another embodiment, the didemnin analog has a fluorophore attached, such as an analog in which a fluorophore is attached at the omega amino moiety of a lysine side chain at R2 or at R10. An example of the structure of such a fluorescent didemnin analog is show in FIG. 29. Alternatively, the didemnin analog can be attached (e.g. covalently) with a support. In most embodiments, Y in formula I is preferably a hydride radical.
The invention includes an embodiment of a didemnin analog which can be activated (or the activity of which can be enhanced) by enzymatic cleavage of a moiety bound with the analog. For example, the invention includes compositions which comprise a didemnin analog having the structure of formula II 
In formula II, R2, R3, R4, R10, X, and Y have the same identities described above for formula I. R13 is an enzyme-cleavable moiety that is cleavable by an enzyme, such as one selected from the group consisting of a carboxypeptidase, a beta-lactamase, a beta-galactosidase, a penicillin V-amidase, a cytosine deaminase, a nitroreductase, a alkaline phosphatase, a beta-glucuronidase, and a catalytic antibody. By way of example, R13 can have the structure of either of formulas V and VI 
Examples of such didemnin analogs that include compound 131 and compound 132.
The invention also relates to compositions which comprise a didemnin fragment having the structure of formula VII 
In formula VII, Y is either a hydride radical or a hydroxyl protecting group, X is either xe2x80x94Oxe2x80x94 or xe2x80x94(NH)xe2x80x94, R4 is either an isoleucine side chain or a valine side chain, and APG is a amine protecting group. R11 can be any of xe2x80x94OH, xe2x80x94NH2, xe2x80x94O(allyl), xe2x80x94O(pentafluorophenyl), and a substituent having the structure of formula VIII 
In formula VIII, R1, R2, R3, and R10 have the same identities described above for formula I, and R12 can be either a hydride radical or a -2-(trimethylsilyl)ethoxycarbonyl moiety.
The didemnin analogs and fragments described herein can be formulated, together with one or more pharmaceutically acceptable carriers, to make pharmaceutical preparations. These preparations can be administered to a mammalian (e.g. human) cell (i.e. either in vitro or in vivo) in order to inhibit protein synthesis, inhibit growth, inhibit proliferation, inhibiting tumorigenesis, or enhance apoptosis in the cell or in one or more tissues of the mammal.
The invention further includes a method of making a didemnin fragment. This method comprises coupling a first reactant having the structure 
and a second reactant having the structure 
to yield a first didemnin fragment having the structure 
In this structure, X is either xe2x80x94Oxe2x80x94 or xe2x80x94(NH)xe2x80x94, APG is an amine protecting group; Y is a hydroxyl protecting group (e.g. a -triisopropylsilyl group), and R4 can be either an isoleucine side chain or a valine side chain. The first didemnin fragment can be hydrolyzed to yield a second didemnin fragment having the structure 
An activator (ACT) can be added to the carbonyl moiety of the second didemnin fragment to yield a third didemnin fragment having the structure 
The third didemnin fragment can be coupled with a third reactant which has the structure 
to yield a fourth didemnin fragment having the structure 
In this structure, R2 and R3 have the identities described above for formula I, APG is an amine protecting group, SEM is a 2-(tnimethylsilyl)ethoxycarbonyl group, and R10 is either a leucine side chain or a lysine side chain.
The invention also relates to a method of making a didemnin analog from the fourth didemnin fragment. This method comprises removing the SEM and CBZ moieties from the fourth didemnin fragment and cyclizing the fragment to yield a first didemnin analog having the following structure. 
The APG group (which can, for example, be a carbobenzyloxy moiety or a tert-butyloxycarbonyl moiety) can be removed from the first didemnin analog to yield a second didemnin analog having the structure 
This second didemnin analog can be coupled with a fourth reagent having the structure 
to yield a third didemnin analog having the structure 
In these structures, R14 is can be one of
xe2x80x94leucine,
xe2x80x94(N-methyl)leucine,
xe2x80x94(N-methyl)leucine-(S)proline,
xe2x80x94(N-methyl)leucine-(S)proline-pyruvate,
xe2x80x94(N-methyl)leucine-(S)proline-(S)lactate,
xe2x80x94(N-methyl)leucine-(S)proline-(S)lactate-(a first fluorophore),
xe2x80x94(N-methyl)leucine-(S)proline-(S)lactate-(S)glutamine-(S)pyroglutamate,
xe2x80x94(N-methyl)leucine-(S)proline-(S)lactate-(S)glutamine-(S)cyclopentanoate,
xe2x80x94(N-methyl)leucine-(S)proline-(S)alanine-(S)leucine-(S)pyroglutamate, and
xe2x80x94(N-methyl)leucine-(S)proline-(N-methyl-S-alanine-(S)leucine-(S)pyroglutamate, or
it can be one of these moieties coupled with an enzyme-cleavable moiety that is cleavable by an enzyme such as one of a carboxypeptidase, a beta-lactamase, a beta-galactosidase, a penicillin V-amidase, a cytosine deaminase, a nitroreductase, an alkaline phosphatase, a beta-glucuronidase, and a catalytic antibody. If Y is a hydroxyl protecting group, then that can be removed from the third didemnin analog (either before or after addition of R14) to yield a fourth didemnin analog having the structure 